In fluorescence imaging, a light source having a broad spectral density distribution, such as a xenon or mercury arc lamp, ensures that many different fluorescent materials can be utilized. Through an exciter filter, the sample is illuminated with only that portion of the spectrum to which the fluorescent dye is sensitive. At the same time, a darker background is achieved, and organic samples that, for example, are destroyed or altered by UV light are protected. An additional barrier filter in the optical path of the fluorescent light ensures enhanced contrast.
One of the advantages of fluorescence microscopy, in which the wavelengths of the excitation light and the detected light are different, over transmission or reflection microscopy, which work with identical wavelengths, lies in the characteristic properties of fluorescent light since, in addition to the radiation intensity, important information about the sample can also be gathered from the fluorescence lifetime, the spectral distribution and the polarization.
In a conventional reflected-light fluorescence microscope, the sample is irradiated with light, and fluorescent dyes with which the sample was stained emit the fluorescent light that the experimenter can observe through the eyepiece. With the aid of a filter, the excitation light that is also still entering the detection optical path is suppressed so that the fluorescence is not outshone.
The reflected-light fluorescence microscope further includes a dichroic splitter mirror, whose task is to reflect the excitation radiation to the sample as completely as possible, and to transmit the fluorescent light to the eyepiece as completely as possible. Thus, its dichroism is selected such that the mirror is transparent for the fluorescence wavelength, and is a mirror for the excitation wavelength. Depending on the optical path, a reverse distribution of transparency and mirroring can also be realized; the splitter mirror then constitutes a mirror for the fluorescence wavelength and is transparent for the excitation wavelength. In both cases, the exciter filter, the dichroic splitter mirror and the barrier filter form a component part that is simply interchanged by swiveling.
In transmitted-light fluorescence microscopes, the requirements for the filter are very high, and it is hardly possible to achieve a dark background.
Dark-field microscopes do not allow any direct microscope light to enter the objective. Here, the sample is not illuminated with a cone of light, but rather only with the envelope of a cone. This cone envelope is prepared by a special condenser, known as a dark-field condenser. If there is no sample in the optical path, the field of view remains completely dark when looking into the eyepiece. If, on the other hand, a specimen is placed in the optical path, the microscope light is partially deflected by the sample structures through refraction, reflection, diffraction or scattering. Some of the light deflected in this way now enters the objective and produces a visible image in the microscope. The objects thus generally appear brightly glowing on a dark background. Here, particularly the edges and scattering interfaces glow; the interior of an object usually remains dark.
In laser scanning microscopes, the sample is analyzed in a scanning method with the aid of a laser beam that scans the sample. The grid-point data measured is subsequently composed into an image of the sample.
The advantage of confocal light microscopy consists in the possibility to collect the light reflected or emitted by the sample from a single depth plane. A pinhole aperture that is conjugate to the focal plane, or in other words, is disposed confocally, ensures that nearly all light that does not originate from the focal plane is not detected by the detector.
In a confocal laser scanning microscope, as with a laser scanning microscope, an image is composed point by point and line by line from a sequential scanning. By shifting the focal plane, the two-dimensional images of a specific depth, the optical cuts, can be composed into a three-dimensional image, and in particular, can be further processed digitally.
However, each of the methods cited exhibits disadvantages. For example, in fluorescence imaging, the required suppression of the excitation light by filters limits the signal-to-noise ratio that can be achieved.
Excitation with a laser scanner offers the benefit of narrow spectral width of the excitation light; however, the collinear optical paths also make good filters necessary. In addition, the lasers utilized are costly and the scanning device requires intensive maintenance and repairs due to the movable parts.
If a CCD element is utilized as the detector, dark-field excitation is usually preferred. Then the signal-to-noise ratio is determined by the inherent noise of the CCD chip, so that in order to achieve higher sensitivities, long integration times are needed and the element must be cooled utilizing a Peltier cooler or liquid nitrogen. This solution is costly, too, due to the utilization of technically sophisticated CCD elements, and their high resolution is not needed and not used for many applications.